KR19990044645A - Toxoplasma gondiga conjugate - Google Patents

Abstract

The present invention relates to a glycoside conjugate having immunoreactivity with Toxoplasma gondii antibody, a (monoclonal) antibody to these glycoside conjugates, and a cell line capable of producing a monoclonal antibody. The present invention is also directed to a process for the preparation of < RTI ID = And to an immunochemical reagent and method for detecting an antibody directed against Gondii. The present invention also relates to a therapeutic agent for inhibiting the replication of the T. gondii organism and a method for producing the attenuated T. gondii live vaccine.

In contrast to what was disclosed in the earliest art, several glycoside conjugates have been shown to be immunoreactive with Toxoplasma gondii specific antibodies comprising a glycan core structure of the formula 1:

Formula 1

In this formula,

R 1 = hydrogen, -PO ₃ -CH ₂ -CH ₂ -NH ₂ (ethanolamine-phosphate) or -PO ₃ -CH ₂ -CH ₂ -NH-X, wherein X = Gondii-specific antigens;

(1-4) -O-anhydrous mannitol or [alpha] (1-4) -O-glucoseamine, optionally in the inositol portion of the phosphatidyl inositol membrane adherend, ;

R3 = monosaccharide moiety;

R4 = hydrogen or -L-R5, L = 2 is an organic radical;

R5 = a functional group capable of coupling to the carrier.

One preferred embodiment of the monosaccharide moiety is a hexose sugar moiety, more preferably a glucose moiety, most preferably a glucose moiety bonded to a GalNAc moiety with an alpha -1,4 glycoside bond. The saccharide conjugate of the present invention comprises It is especially suitable for diagnosing Gondy infected human body.

Description

Toxoplasma gondiga conjugate

Toxoplasma gondii is an intracellular prototropic organism found worldwide and is capable of infecting all mammalian species and all species belonging to a given individual. T. gonadis are classified into two life cycles, that is, staphylococci having a silent and oily phase.

In the silent phase, T. gondi exists in many forms. For example: tachyzoites, cauliflower, bradyjot, and cysts. Takijito is an acute infection. It is a form of gonadal knockout cells. For human host cells. The infectious stage of Gondi is caustic. A cauliflower has a diameter of 30 to 100 micrometers and includes hundreds to several thousands of infectious units called bladizoids. Frequent ingestion of pseudo-pods present in raw or uncooked non-frozen meat will trigger the infection. In addition, infections may occur due to ingestion of cysts on the sides of cats that have been infected with active intestinal infections. It can also be transmitted through blood or white blood cell transfusion by organ transplantation or light placental transfer during pregnancy (Wilson et al., J. Exp. Med. 151, 328-346, 1980). The wall of the caudate or cyst is decomposed in the small intestine by the host digestive enzymes, releasing the bradyzoites or sporozoites, respectively, and then infiltrating the sooty epithelial cells. Bradyzoites or sporozoites reach the liver by the blood-source pathway, where they are ingested by Kupffer cells. Once inside the cell, this organism is called Takijito. Liver Membrane Cells are also infected (Kranenbuhl & Remington, Immun. Of Parasitic Infections; eds. Cohen, S., Warren, KS, London: Blackwell Scientific Publications p356-421, 1982; Remington & Kranenbuhl, Immun. II, edited by Nahmias, AJ, O'Reilly, RJ New York, Plenum Medical Book Company, p. 327-371, 1982).

It is replicated immediately after transfer into the host cell. In the host, macrophages deliver Brady's to the entire body. Brady < / RTI > jets survive and replicate in the vacuole by preventing the fusion of macrophage biocompatible vacuole and lysosome. As a result of replication, host cells are lysed. New macrophages or other cell types phagocytose the organism, and this cycle is repeated.

tea. The oily phase of Gondii occurs only in feline hosts (Miller et al., J. Parasitology, 58, p928-937, 1972). Intermediate hosts (eg, mice) are infected by ingesting cysts or caudal cannulas. The caudal cannula is distributed throughout the whole tissue of the mouse. Cats are infected when they eat infected meat (eg, rodent tissue) that contains caustic or when they ingest cysts. Brady < / RTI > joules or sporozoites infiltrate into a soju-like epithelial cell and differentiate into schistosomes. After cloning, cleavage bodies rupture the infected epithelial cells and infect neighbors. Some schizoid bodies are differentiated into pre-somatic cells called large-spontaneous (female) and bovine spontaneous (male). Bovine mother cells fuse with large spontaneous cells to form a zygote, called a cyst (Dubey & Frenkel, J. Protozool. 19: 155-177, 1972). The cyst enters the lumen of the small intestine and is defecated. Each cyst forms spores that produce eight infectious sporozoites in the soil, which is the infectious stage of the intermediate host.

Brady < / RTI > zites are present in the form of chronic and potential infections wrapped in cysts for about 8-10 days after in vivo entry. Thus, the Brady's form is the only step that can initiate the intestinal epithelial cell cycle (Krahenbuhl & Remington, 1982; Remington & Krahenbuhl, 1982).

tea. Gondii causes mild or asymptomatic disease in healthy adults, but it causes serious, toxoplasmosis, or even death in congenital infected children and immunocompromised patients. Primary infections of pregnant women occur in Europe with a frequency of 0.2 to 1.0%. In this case, about 40 to 50% of fetuses are infected. Infection of the fetus during pregnancy results in a child with neonatal death or severe multiple disorder (approximately 10% in this case), but 90% of the cases are born with a child asymptomatic, latent infectious agent (Desmonts and Couvreur, Ann Pediatr 1984, 31, 805-809; Alford et al., Bull. NY Acad. Med. 1974, 50, 160-181). Up to 85% of patients with latent congenital toxoplasmosis will form significant sequelae involving one or more episodes of active chorioretinitis. Other clinical symptoms are inflammation, lymph nodes, encephalitis and fever.

Congenital toxoplasmosis is caused by nucleic acid amplification technology. Gondii IgM and / or IgA antibodies or T. (Burg et al., J. Clinical Microbiol., 27, 1787-1792, 1989; Savva et al., J. Med. Microbiol., 32, 25-31, 1990).

Several known tees. There are Gondii strains. One of the most important strains is the RH strain, which is extremely toxic and originally isolated from human brain tissue.

tea. Most of the past investigations on Gondii have focused on the identification and molecular characterization of Tachyzoites-specific antigens. Over 1000 different tees. Gondi-specific proteins were identified. Because the membrane-adherent surface proteins and secreted / excreted proteins of T. gondii are highly immunogenic, they have been intensively studied. Of the surface proteins, p30 is the most abundant, It constitutes about 5% of the total TKI protein of the Gondii RH strain. p30 protein is intensively recognized by human IgM, IgG, IgA and IgE antibodies (Decoster et al., Clin. Exp. Immunol., 73, 376-382, 1988; Godard et al., Infect. Immun., 58, 2446-2450 , 1990) and are therefore useful for diagnostic purposes. (Cesbron et al., J. Immunol. Methods 83, 151-158, 1985) and IgA (Decoster et al., Lancet, ii, 1104-1106, 1988) using monoclonal antibodies directed against p30 Two immunoassay tests were developed to detect antibodies. Another tea with a molecular weight of 4-10 kDa. Immunopotential antigens of Gondi were identified. The 4-10 kDa antigen is predominantly recognized by human IgM and is recognized in a smaller extent by human IgG antibodies (Sharma et al., J. Immunol. 131: 977-983, 1983). In addition, treatment of the membrane component with NaIO4, an agent known to oxidize the adjacent hydroxyl group of the sugar moiety, can inhibit the IgM and IgM antibodies in the ELISA as well as the IgG antibody. Immunoreactivity of Gondii antigens has been observed to be significantly reduced (Naot et al., Infect. Immun. 41, 331-338, 1983). Sharma et al. (J. Immunol. 131: 977-983, 1983) showed that membrane-enriched fractions isolated from NaIO4-treated Toxoplasma ultrasound or SDS gel exhibited high affinity for human IgM and IgG antibodies and immunostaining. It was observed that the immunoreactivity of Gondii antigen was abruptly decreased.

Especially, Immunoreactivity of Gondii antibody and 4-10 kDa antigen was completely destroyed after NaIO4 treatment. In addition, the 4-10 kDa molecule is not responsive to the protease and trypsin treatment, but is sensitive to lipases that display the structure of the glycolipid structure. Briefly, in the above-mentioned study, It can be seen that the Gondi carbohydrate molecule plays a role in inducing an effective immune response in the infected host.

Tissue biology of glycosyl phosphatidylinositol (GPI)

It is widely known that deformation after cleavage of proteins by oligosaccharides and glycolipids plays an important role in the structure, biosynthesis, and biological functions of surface membrane proteins of viral, bacterial and eukaryotic cells, including parasitic protozoa.

Proteins are anchored to the membrane through covalent bonds to the glycosyl-phosphatidylinositol (GPI) molecule through expansion of hydrophobic amino acids that span the lipid bilayer (epidermis) or during translocation of the developing protein into the lumen of the rough endoplasmic reticulum. In all reported cases, lipid anchors replace short sequences of primarily hydrophobic amino acids at the carboxyl terminus of the protein, and thus are considered another attachment mechanism for the dermal polypeptide domain of form-1 membrane proteins (Ferguson and Williams, Ann. Rev. Biochem., 57, 285-320, 1988; Ferguson, Curr Opinion in Struct Biol., 1, 522, 1991).

Most information on the structure and biosynthesis of the saccharide membrane fixtures was obtained through studies of the modified surface glycoprotein (VSG) and GPI of the parasitic protoplasm, Trypanosoma brucei. The glycolipid fixation of a VSG (VSG117) is chemically defined and consists of the framework sequence of ethanolamine-phosphate-6Manα1-2Manα1-6Manα1-4GlcNα1-6-inositol. The inositol residue is linked to dimyristoyl glycerol via a phosphodiester bond, the ethanolamine group is amide-linked to the carboxy terminal amino acid of the mature protein, and the galactose side chain is attached to the mannose residue adjacent to glycosamin. Similar skeletal structures with different side chain modifications and lipophilic groups have been described for lipid attachment of some other proteins (see Ferguson and Williams, Ann. Rev. Biochem. 57: 285-320, 1988; Cell Biol 6: 1-39, 1990; Ferguson, Curr Opinion in Structural Biology 1, 522-529, 1991).

tea. Gondis glycosyl phosphatidylinositol fixture

Until recently, little is known about the glycosylation of Toxoplasma gondii and there was considerable discussion of the presence of glycoproteins as the major surface component of toxoplasma. Handman et al. (J. Immunol. 124, 2578-2583, 1980) found no difference in the composition of the membrane antigen even after passage through radioactive iodine-treated guar gum in the ConA-Sepharose column . In contrast, using a radioactively labeled ConA, Johnson et al (Biochem. Biophys. Res. Commun. 100, 934-943, 1981) and Mauras et al. (Biochem. Biophys. Res. Commun. 97, 906-912, 1980) The binding of lectin to toxoplasma was detected.

It is of fundamental importance to compare the GPIs bound to the putative precursor molecule and the protein to establish a detailed biosynthetic pathway. The data presented by Schwarz et al. (NATO ASI Series. Series H: Cell Biology, vol. 78, Berlin, Springer, 1993. p109-121) The p23 of the gonadotachyzoites contains a skeleton with a high degree of conservatism similar to that of the mammalian Thy-1 anchor, which skeleton contains a modified version of the ethanolamine-PO4-6Manα1-2Manα1 branched side chain identified as the N-acetyl-galactosamine residue (See Ferguson et al., Science 239: 753-759, 1988; Zinecker et al., Diplomarbeit, University of Marburg, 1992).

Similar assays were performed by comparable analysis of p30 (Tomavo et al., J. Biol. Chem. 267: 11721-11728, 1992). Thus, the attachment of the two proteins is the same for carbohydrates. Prove some micro-heterogeneity in glycan of p30 (Zinecker et al., 1992) and examine whether this is a common property of Toxoplasma GPI-membrane fixation.

tea. Because the antibody induced for GPI fixation of Gondii does not bind to GPI fixation of other protoplasts, Gondi's GPI fixation is expected to have a unique component in addition to the more basic ethanolamine-P-6Manα1-2Manα1-6Manα1-4GlcNα1-6inositol core structure.

Therefore, it is important to elucidate the structure of GPI-membrane anchorage for glycans and lipid moieties and to find structural features that are not found in mammalian cells.

tea. When the main surface protein of Gondii is fixed to the surface of the organism by GPI-fixing (Tomavo et al., Mol. Cell. Biol. 9: 4576-4580, 1989; Nagel et al., J. Biol. Chem. 264: 5569-5574 , 1989), these membrane anchoring and in particular the biological function and biosynthesis of the putative precursor are important in the art. The observation that the highly immunogenic 4-10 kDa antigen is a precursor species of the unconjugated or free glycosyl phosphatidylinositol fixate is shown in Fig. To elucidate the biochemical structure and mapping of the epitope (s) to Gondi's GPI fixation. tea. Information on the structure and immuno-dominant epitope of Gondi's GPI fixation allows chemical synthesis and application of artificial GPI fixtures in diagnostic assays. In contrast to native GPI fixtures, artificial carbohydrate molecules analogous to immunoreactive sites can be chemically modified to be directly coupled to the enzyme conjugate by linker molecules.

Therefore, In order to develop a specific and sensitive method for forming a reliable diagnostic method for Gonadal infectious diseases, it is important to identify immunogenic dominant glycoconjugates that serve to accurately bind specific antigen.

tea. By understanding the structure of Gondi GPI fixation, it is possible to inhibit or inhibit the action / activity of the intrinsic enzyme to inhibit the biosynthesis and binding of a unique carbohydrate substituent to the GPI core structure. This is T. The specific inhibition or blocking of GPI fixation synthesis of Gondii reduces the cell surface expression of the protein and can ultimately be fatal to the parasite. For example, T. Brussey's GPI precursor is modified for fatty acids. These modifications are unique to tripopanos and are an excellent target for the development of rheumotherapeutics, such as myristate analog 10- (propoxy) decanoic acid, which exhibit selective toxicity to tripopanos (Doering et al. Science 252, 1851-1854, 1991; Masterson et al., Cell 62, 73-80, 1990).

The role of GPI in the biology and pathology of parasites is described in detail. However, by identifying the structure of the GPI-fixture, a novel approach to the development of anti-toxoplasma drugs based on the damage of the bio-specific carbohydrate structure will be possible.

The primary function of the GPI fixture is to give the plasma membrane or, in some cases, a stable relationship to the lumen surface and protein of the splenic. The feature of GPI fasteners is that T. Can be used to express a heterologous gene encoding a protein on the surface of Gondii. Several studies have demonstrated that the efficiency of membrane targeting and turnover is determined solely by GPI fixtures. Therefore, T. By identifying the structure of Gondi's GPI fixation, it is possible to transform the heterologous gene in a way that effectively binds to the GPI fixation, and membrane targeting is possible through GPI fixation. Ultimately, as a heterologous gene expression system, With Gondi, an efficient and reliable vaccine development approach can be obtained. In addition, GPIs attached to proteins are stable in binding to the membrane, but can be more easily dissolved in detergents than in membrane proteins (Hooper and Turner, Biochem. J. 250, 865-869, 1988). As a heterologous gene expression system for expressing protein chimeras with GPI fixation, With Gondi, a reliable antigen source can be easily and constantly increased and the expressed heterologous protein can be easily extracted with a detergent (CHAPS, Triton X-114).

Experimental results using B-cell depletion (IgM-suppressed) BALB / C mice suggest that anti-toxoplasma antibodies are essential for the regulation of long-term toxoplasmosis, although not essential for the recovery of acute infection Frenkel and Taylor, Infection & Immunity, 38, 360-367, 1982). This data shows that the glycolipid structure is a T-helper cell-dependent B cell mitogen, which in turn leads to B cells that produce IgM antibodies. Therefore, Detailed information on Gondi's GPI fixture structure. An artificial GPI anchoring molecule can be synthesized that binds to an appropriate ligand that can be applied as a mitogen for inducing a protective antibody response in a susceptible host of Gondii.

The present invention relates to a glycoside conjugate having immunoreactivity with Toxoplasma gondii (hereinafter referred to as T. gondii) antibody, a (monoclonal) antibody against these conjugates, and a cell line capable of producing a monoclonal antibody . The present invention is also directed to a process for the preparation of < RTI ID = And to immunochemical reagents and methods for detecting antibodies directed against Gondii. The present invention also relates to a therapeutic agent for inhibiting the replication of the T. gondii organism and a method for producing the attenuated T. gondii live vaccine.

Figure 1: Antigenic specificity of monoclonal antibodies and polyclonal antibodies as evidenced by western blot analysis of total solids in which T. gondii was dissolved . That is, the filters were treated with two human anti-toxoplasma IgM monoclonal antibodies (lanes 1 and 2, respectively) directed against glycolipid TOX.HuOT-1 and TOX.HuOT-2, two human anti-toxoplasma IgM monoclonal, TOX.HuOT-3 and TOX.HuOT-8 (lanes 3 and 4), mouse anti-glycolipid monoclonal antibodies T54E10 and T33F12 (lanes 5 and 6 respectively), mouse anti-human chorionic gonadal Hormone (lane 7) and a collection of five human anti-toxoplasma IgM fractions (lane 8) as a positive control.

Figure 2: Western blot analysis of T. gonadal gelatin (butanol phase): Human serum transduction serum IgM (A) and IgG (B) and mouse monoclonal antibodies T33F12 (C) and T54E10 (D) (Tomavo et al. Parasitol, 108: 139-145, 1994).

Figure 3: T TLC analysis of glycolipids gondi: ³ H- glucosamine (A), ³ H- palmitic acid (B), ³ H- mannose (C) and ³ H- ethanolamine (D) the metabolically labeled with Butanol phase derived from organism.

Figure 4: Point blot analysis of TLC purified T. gonadal glycolipids: TLC purified glycolipids were spotted onto nitrocellulose and then reacted with a collection of five human α-Tox IgM fractions and mouse α-glycolipid mAb T54E10.

Figure 5: TLC purification. Point blot analysis of T. gonadal glycolipids: TLC purified glycolipids were spotted onto nitrocellulose and reacted with human serum-transformed serum.

Figure 6: Inhibitory ELISA: Inhibitory ELISA using the glycolipid and the mAb T54E10 contained on butanol as antigen.

Figure 7: Analysis of core glycans from TLC purified glycolipids labeled with H-glucosamine on agarose of Wistaria floribunda agglutination agarose: Coreglycan A (A), coreglycan (B), rat brain Of Man ₄ GalNAcAHM (C) from the Thy1 anchor, and the arrows show buffer changes.

Figure 8: Dionex-HPAEC and Aminopropyl-HPLC analysis of core glycans from labeled glycolipids in vitro using Tachyzoite cell-free system: Left column shows HPAEC-HPCL analysis, right column shows Aminopropyl-HPLC (A, B): In vivo labeled core glycans, unlabeled UDP-Glc members (C, D) and presence (E, F) And G and F represent core glycans derived from glycolipids labeled in vitro with UDP-3H-Glc.

Figure 9: Analysis of HPAEC monosaccharide labeled in vitro with HPLC-purified coreglycan B via UDP-3H-Glc: Coreglycan B was hydrolyzed with 2N TFA for 4 hours at 100 < 0 & And analyzed by HPAEC eluted with NaOH.

10: Direct purification of core glycans from the butanol phase using purification HPLC: Core glycol (from butanol) derived from glycolipids labeled with ³ H-glucosamine was dissolved in Lichrosorb NH ₂ (A), equivalent materials (B, C ) And fractions collected under peaks A and B (D, E and F, G, respectively) were analyzed with HPAEC (left column) and bio gel P4 (right column), with Arabic numerals indicating the position of the standard internal dextran hydrolyzate .

Figure 11: GC-monosaccharide analysis of core glycan samples: HPLC fractions containing core glycans A (A) and B (B) were subjected to methanolysis, re-N- acetylation and TMS- derivatization and analyzed by GC Respectively.

Figure 12: Fast atom impact mass spectrometry (FAB-MS) of core glycans : HPLC Purified core glycans A (A) and B (B) were hypermethylated and analyzed by FAB-MS.

Figure 13: Core glycans A and B at 500 mHz ^One H-NMR spectrum

14: methylation analysis of core glycan B : 80 nmol of Coreglycan B was hypermethylated, hydrolyzed, reduced and acetylated, and the resulting PMMA was analyzed by GC-MS. This figure shows the total ion chromatogram.

15: A schematic diagram of a conjugate structure of T. gondii. This structure shows the sugar conjugate disclosed in the present invention. The bioconjugates may vary depending on the presence or absence of the terminal ethanolamine phosphate and / or the lipid moieties thereof.

Figure 16: Representative view of the T. gondii conjugate structure. This structure shows the sugar conjugate disclosed in the present invention, wherein A represents a core glycan without a unique glucose residue and B represents a core glycan with a unique glucose residue. The R1 and R2 groups are as described herein. The bioconjugates may vary depending on the presence or absence of the terminal ethanolamine phosphate and / or the lipid moieties thereof.

17: Reaction scheme. In this reaction scheme, the synthesis and conjugation of the sugar conjugate disclosed in the present invention is shown in detail in accordance with Example 8.

In contrast to earlier publications in the art, it has been found that some glycoside conjugates are immunoreactive with toxoplasma gondii-specific antibodies comprising a glycan core structure of formula 1:

In this formula,

R 1 = hydrogen, -PO ₃ -CH ₂ -CH ₂ -NH ₂ (ethanolamine-phosphate) or -PO ₃ -CH ₂ -CH ₂ -NH-X, wherein X = Is a Gondii specific antigen;

(1-4) -O-anhydrous mannitol or [alpha] (1-4) -O-glucoseamine, optionally with an alpha (1-6) -linkage to the inositol moiety of a phosphatidyl inositol membrane adherend ;

R3 = monosaccharide moiety;

R4 = hydrogen or -L-R5, L = 2 is an organic radical;

R5 = a functional group capable of coupling to the carrier.

The term "divalent organic radical" of the present invention generally means a linker structure in which the functional group R ₅ is bonded to the glycan core. Suitable divalent organic radicals are, for example, substituted or unsubstituted radicals such as methylene [-CH ₂ -], ethylene [-CH ₂ -CH ₂ -], and preferably propylene [-CH ₂ -CH ₂ -CH ₂ - An alkylene group or an aralkylene group.

The term " functional group capable of coupling to the carrier " of the present invention generally includes any reactive functional group that can be used to covalently couple the inventive glycan core structure to the carrier, ≪ / RTI > Specific functional groups are mercapto (-SH), amino (-NH ₂ ), pyridyldithio, S-acetyl-mercaptoacetyl (SATA), maleimido, carboxylic acid functional groups or esters thereof, Esters derived from N-hydroxysuccinimide, p-nitrophenol or pentafluorophenol, bromoacetamido, vinylpyridine, hydrazide, aldehyde or hydroxy. Additional suitable functional groups are those known in the art of bonding chemistry.

&Quot; Carrier " of the present invention generally means a solid support that can be used for any substance to which the glycan core structure of the present invention can be conjugated, such as a protein (such as albumin or keyhole lipopeptide hemocyanin) and diagnostic reagents.

A preferred first aspect of the present invention is a sugar conjugate wherein the monosaccharide portion is a saccharide sugar.

In a second preferred embodiment of the present invention, said hexose sugar is a glucose sugar conjugate.

A third preferred embodiment of the present invention is a glycoside conjugate wherein a glucose moiety is bonded to a GalNAc moiety by an? -1,4-glycoside bond.

A fourth preferred embodiment of the present invention is a sugar conjugate wherein the sugar conjugate comprises the structure shown in Fig.

A fifth preferred embodiment of the present invention relates to a method of producing a t- And a step of rinsing the Kondi tachyzoite membrane and extracting a Tachyzoite suspension produced with a mixture of chloroform / methanol / water (C / M / W: 1/1 / 0.3 v / v) It is a sugar conjugate obtainable from Gondigakyi zeit.

A preferred sixth aspect of the present invention is a glycoside conjugate immunoreactive with a human monoclonal antibody formed by an infinite proliferating cell line deposited at Accession No. 95090608 and 95090609 to the European animal cell culture collection (ECACC) of Fortton Down, UK .

Glucose conjugates are generic terms used to describe molecules in which carbohydrate moieties (monosaccharides, oligosaccharides, or polysaccharides) are covalently bound to other (living) molecules, such as lipids or polypeptides. However, it is believed that the carbohydrate core structure is also within the scope of the sugar conjugate.

Oligosaccharides (alkyl / acylglycerols, diacylglycerols, sphingosines, ceramides, or dorichol) bound to lipids are referred to as glycolipids (IUB-IUPAC recommendation, 1976, (1976), J. Lipid Res. 19, 114). Glycoproteins are polypeptides in which the oligosaccharide is linked via N- and / or O-glycosidic bonds, and the carbohydrate of the polypeptide may represent 2-60% of the total weight (Montreuil et al., 1982, Comprehensive Biochemistry, vol. 19B , part II, p 1, Florkin, G plate). The oligosaccharide moieties present in these polypeptides are discrete, specific and conserved structures. This property is distinguished from other glycoside conjugates such as proteoglycans, glycosaminoglycans, mucoproteins and peptidoglycans, and amino acids are linked to a carbohydrate chain composed of a variable number of repeating structural units (IUB-IUPAC recommendation, 1980, (1982) J. Biol. Chem. 257, 3352).

Another class of glycosylated conjugates is glycosyl phosphatidylinositol (GPI) lipid anchors (Low et al., 1986, Trends Biochem. Sci. 11, 212). They link cell surface glycoproteins to membrane lipids through oligosaccharides. Thus, the glycoprotein can be glycosylated with N- and O-linked oligosaccharides, as well as GPI-fixates (Ferguson et al., 1988, Science 239, 753-759).

The saccharide conjugate can be isolated and purified from a natural source (e.g., from the T. gondii organism in the present invention, preferably from T. gonditaci zeeit). The separation process disclosed in the examples can be used. However, other separation processes known in the art can be used and thus fall within the scope of the present invention.

Another method for obtaining the sugar conjugate of the present invention is a chemical synthesis pathway using a method often used in the art. The synthesis route is illustrated in one embodiment below. However, other synthetic routes known in the art are within the scope of the present invention as they are considered equivalent.

Antibodies derived for the sugar conjugate of the present invention are also part of the present invention.

The sugar conjugate prepared as described above can be used to generate antibodies (polyclonal and monoclonal). Monoclonal antibodies directed against the glycoside conjugate of the present invention can be readily produced by those skilled in the art.

Accordingly, the monoclonal antibody of the present invention is a monoclonal antibody. Provides a new method for the diagnosis of Gonadal infections.

A preferred antibody of the present invention is T. Monoclonal antibody that binds to the epitope of the gonadal < RTI ID = 0.0 > conjugate, < / RTI > which epitope is a monoclonal antibody produced by a hybridoma cell line deposited with accession numbers 95090608 and 95090609, respectively, in the European animal cell culture collection (ECACC) It is recognized by the LOHAL antibody TOX.HuOT-1 or TOX-HuOT-2.

Preferred antibodies of the invention are monoclonal antibody TOX.HuOT-1 or TOX-HUOT-1 produced by hybridoma cell lines deposited at Accession Nos. 95090608 and 95090609, respectively, in the European animal cell culture harvester (ECACC) HuOT-2.

The infinitely proliferating cell line capable of secreting the monoclonal antibody of the present invention is also part of the present invention.

Cell lines that generate monoclonal antibodies have been described, for example, in Kohler and Milstein techniques (Kohler and Milstein), which devised a technique for forming monoclonal antibody-producing hybridomas (G. Kohler and C. Milstein, 1975, Nature 256 Transfection with Epstein-Barr virus, direct transformation of B-lymphocytes using tumorigenic DNA, human or mouse-human Direct fusion of the hybrid myeloma cell line fusion partner with human B-lymphocytes, or direct fusion of the EBV-transformed B cell line with the myeloma cell line.

A preferred cell line of the present invention is a cell line deposited at Accession No. 95090608 and 95090609, respectively, in the European animal cell culture collection (ECACC), Fortton Down, UK.

These hybridoma cell lines were prepared using adjuvant Epstein Barvirus (EBV). EBV can transform and infinitely proliferate human B-lymphocytes. Infinitely proliferated human B-lymphocytes can be obtained with the aid of EBV without myeloma partner cells. Epstein barbitus can be obtained from a variety of sources. The most common used as a source of EBV is the B95-8 mamozet cell line. The B95-8 cell line spontaneously releases EBV into the medium.

tea. The monoclonal antibody to the glycosidic conjugate of Gondii is useful for in vitro and in vivo cell and cell extracts. Is a useful means of detecting the expression of Gondii and is for various biochemical and immunological analytical techniques for studying the purification purpose and the action of proteins.

Immunochemical reagents comprising the present conjugate or antibody of the invention are also part of the present invention.

&Quot; Immunochemical reagents " of the present invention generally include one or more of the present conjugates of the invention and a suitable support or labeling material.

Supports that can be used include, for example, the inner wall of a microspheroidal plate well or cube, a tube or capillary, a membrane, a filter, a test strip or latex particle, an aldehyde particle (e.g., a ceramic magnetizable particle with an active aldehyde surface group) Metal sols such as dye sols, metal sols or sol particles, and carrier proteins such as BSA or KLH.

Available labeling substances are radioisotopes, fluorescent compounds, enzymes, dye sols, metal compounds of metal sol or sol particles.

In the sample, In a method for detecting an antibody directed against a Gondii, the immunochemical reagent of the present invention is contacted with a sample. Thereafter, the presence of an immunocomplex formed between the sugar conjugate and the antibody in the sample was detected, and by this detection, The presence of Gondii antibodies can be known and quantitatively measured.

Depending on the nature and further characteristics of the immunochemical reagent, the resulting immunochemical reaction is called a sandwich reaction, adhesion reaction, competitive reaction or inhibition reaction.

In the sample, In order to detect the Gondii, the immunochemical reagent containing the sugar conjugate of the present invention is used as a sample and anti-T. The presence of the immunocomplex formed after contact with the Gordian antibody was detected and from this, The presence of Gondi can be determined.

In the sample, Particularly suitable methods for detecting the Gondii include the sugar conjugate of the present invention provided as a labeling substance, On the basis of the competition reaction between the Gondi antigen (present in the sample), the sugar conjugate and the antigen are attached to the solid support. And compete with antibodies directed against Gondii.

The present invention also relates to a method for screening an antibody of the present invention, Wherein the presence of the immunocomplex formed to determine the presence of Gondii is detected. And a method of detecting gondii.

The test kit of the present invention comprises the above-mentioned immunochemical reagent as an essential component. tea. In order to carry out a sandwich reaction to detect a Gondii antibody, the test kit may comprise, for example, a sugar conjugate of the invention coated on a solid support (e. G., The inner wall of a microscopic assay plate well), and a labeled sugar conjugate of the invention And may include labeled anti-antibodies.

To perform a competitive reaction, the test kit comprises a sugar conjugate coated on a solid support, And a monoclonal antibody directed against the Gonad, preferably a monoclonal antibody directed against the glycoside.

In a coalescence reaction, the test kit comprises an immunochemical reagent which may comprise a sugar or conjugate of the invention coated on a particle or sol.

In another aspect, the test kit may include, for example, a test kit coated on a solid support. In order to detect the binding site of the antibody phase induced against Gondii. The labeled sugar conjugate of the present invention is used as an immunochemical reagent in a competitive reaction with the Gondii antigen.

The present invention also relates to a process for the production of The therapeutic agent includes a specific inhibitor that inhibits the biosynthesis of the sugar conjugate of the present invention. Examples of specific inhibitors include, but are not limited to, Is a compound capable of inhibiting an enzyme which is specific and necessary for the introduction of the glucose moiety of the conjugate of the Gondigans. This compound may be a chemical compound, and other compounds known to be capable of inhibiting specific enzymes may also be used. GPI-fixate synthesis can be inhibited by genetic manipulation, thereby eliminating genes encoding enzymes involved in GPI-fixate synthesis ("knock-out" mutants). This can be done at the level of transcription or decryption.

The present invention also relates to a method of reducing attenuated tea. Which method comprises blocking or suppressing surface expression of GPI-fixed antigens by the therapeutic agent of the present invention described above. The inhibition or blocking of the surface expression of GPI-fixed antigens is inhibited by T. Reduces the toxicity of Gondii. In addition, " knock-out " variants of so-called GPI-fixture synthesis can be applied as attenuated live vaccines.

A prophylactic vaccine against toxoplasma gondii is also part of the present invention. These vaccines comprise a glycoside conjugate of the invention, together with a pharmaceutically acceptable carrier, including cleavage promoting properties.

In some cases, the ability to increase prophylactic immunity using these conjugates may be low. To increase its immunogenicity, it is preferable to conjugate these sugar conjugates to carrier molecules. Suitable carriers for this purpose include, but are not limited to, macromolecules such as natural polymers (e.g., keyhole limpet hemocyanin, albumin, toxins), synthetic polymers such as polyamino acids (polylysine, polyalanine), or micelles of both affinity compounds such as saponin It is a molecule.

The vaccine of the present invention may be administered according to conventional active immunization schedules: in a manner compatible with the dosing agent, a therapeutically effective amount (i. E., Inducing immunity to the administration of the toxin plasma gonadal biopsy organism Or an amount of the recombinant microorganism capable of expressing the antigen). Immunity is defined as causing a significant degree of prevention in populations after vaccination compared to non-vaccinated groups.

Administration of the vaccine can be done, for example, intradermally, subcutaneously, intramuscularly, intraperitoneally, intravenously, orally or intranasally.

Vaccines may also include suspensions, for example aqueous media or water, often mixed with other ingredients to prolong the duration of the active and / or storage. These components may be selected from the group consisting of salts, pH buffers, stabilizers (skimmed milk or casein hydrolysates), emulsifiers, adjuvants that enhance the immune response (eg, oils, muramyldipeptides, aluminum hydroxides, saponins, polyanions, ) And a preservative.

The present invention is explained in detail through the following examples.

Example 1

Mass production of Toxoplasma gondii takijoites

The RH strain of Toxoplasma gondii (hereinafter referred to as T. gondii) is the most commonly used laboratory strain for toxoplasmosis research. Mass production of T. gonditaci zoyts was carried out by infecting a cell factory (6000 cm 2) containing Vero cells with the above-mentioned Takijyot with multiple infections of 0.30 to 1.0. After incubation at 37 ° C for 3 days, the medium was removed and the cells were reinfused with fresh medium. On the 6th day after infection, the culture supernatant was collected, and the remaining Vero cells were washed with 200 ml of phosphate buffered saline (PBS). The supernatant was collected and the tachyzoite was filtered on a 10 탆 polycarbonate membrane (Nucleopore, Pleasanton, CA, USA) and washed twice with PBS. Tachyzoites were submerged (1,200 x g, 5 minutes) and stored at -70 ° C.

Metabolic labeling of takizoites has been largely described by Tomavo et al. 1992, JBC, 267, 11721-11728. Briefly, the fused monolayer of Vero cells (250 cm 2) is infected with 5 x 10 ⁷ Takijyto added in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2% fetal calf serum. 72 hours after infection, cell cultures were washed twice with glucose-depleted DMEM containing 20 mM sodium pyruvate. The labeling was carried out at 37 ° C. for 4 hours in the same medium as above supplemented with 0.5 mCi [ ³ H] glucosamine. After labeling, it was rotated 20 times in a Dounce homogenizer to dissociate the organism from the host cells. Takizoites were purified by glass wool filtration as described in the literature (Grimwood et al., Exp.Parasitol. 48, 282-286, 1979).

Example 2

Immunoreactivity of the conjugate

Immunoreactivity of the glycosidic bond, more specifically of the GPI molecule, to the human anti-toxoplasma IgM antibody was demonstrated by analysis of purified tachyzoites on SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and western blot . First, the binding sites on the nitrocellulose foliage were blocked by incubating the fleece in a phosphate buffered saline (PBS) solution supplemented with 20% fetal calf serum for 1 hour at room temperature. The fleece strips were then incubated with a human anti-toxoplasma IgM monoclonal antibody directed against the 4-10 kDa fractions of T. gondii as previously reported (Sharma et al., J. Immunol. 131, 977-983, 1983) Lt; / RTI > In addition, five human anti-toxoplasma IgM fraction collections obtained by affinity chromatography using protein G Sepharose 4 Fast Flow (LKB Pharmacia, Uppsala, Sweden) as ligands were evaluated. That is, after incubation at room temperature for 2 hours, the strips were washed with phosphate buffered saline Tween ^R (PBST) and incubated with alkaline phosphatase conjugated goat anti-human IgM antibody for 1 hour. The filter strips were washed and 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (BCIP / NBT, Promega Corp. product of Madison, Wis.) Were added to express the immunocomplex. TOX.HuOT-1 and TOX.HuOT-2 all recognized anti-toxin plasma IgM monoclonal antibodies (FIG. 1, lanes 1 and 2). The collected IgM antibodies recognize multiple T. gondii antigens whereas human IgM monoclonal antibodies do not recognize GPI molecules bound to T. gondii proteins such as the immunopotent antigen p30. In addition to the monoclonal antibodies TOX.HuOT-1 and TOX.HuOT-2, two different monoclonal IgM antibody-producing cell lines were isolated from the same T. gondii infected patient. These IgM antibodies, designated TOX.HuOT-3 and TOX.HuOT-8, reacted with Toxoplasma antigen on a Toxonostika IgM ELISA (Orgalon Technica, Inc., Belgium), but the GPI molecule on the Western blot strip (Fig. 1, lanes 3 and 4).

In addition, the two mouse monoclonal antibodies, designated mAb T54E10 and mAb T33F12, are directed against the glycolipid fraction of T. gondii (Fig. 1, lanes 5 and 6). Both human and mouse monoclonal antibodies directed against glycolipids recognize only non-binding GPI molecules. For example, GPI-molecules bound to T. gondii proteins such as p30 are not recognized at all by the monoclonal antibodies tested. Mouse anti-human chorionic gonadotropin (lane 7) and a collection of five human anti-toxoplasma IgM fractions (lane 8) were used as negative and positive controls, respectively.

This data shows that both the mouse antibody epitope (s) as well as the human IgM epitope (s) for the GPI molecule are no longer accessible due to steric hindrance when GPI is covalently linked to the α-COOH group of the final amino acid of the protein by an amide bond Or disruption by protein-ethanolamine crosslinking formation.

Example 3

Identification of GPI species

T. gondigachyjote is a synthetic example of ^3- H-glucosamine, ³ H-mannose, ³ H-palmitoic acid, ³ H-myristic acid, ³ H-stearic acid and ³ H- Chem., 267, 21446-21458, 1992), as described above. ≪ tb >< TABLE > The glycolipids were separated according to conventionally known extraction procedures (Menon et al., EMBO. J., 9, 4249-4258, 1990; Tomavo et al., 1992). Briefly, tachyzoites were extracted twice with chloroform / methanol (CM; 2: 1) to remove neutral lipids, phospholipids and less polar lipids. The CM-residual pellet was then extracted twice with chloroform / methanol / water (CMW; 10: 10: 3) to obtain lipids of greater polarity. The CMW-extracted glycolipids were pooled and purified with water and water-saturated n-butanol. The immunoreactivity of the n-butanol fraction was analyzed after electrophoretic migration to SDS-PAGE and nitrocellulose membranes using human murine monoclonal antibodies T33F12 and T54E10 as well as human IgM and IgG antibodies containing serum samples. These studies have shown that human anti-toxoplasma IgM antibody strongly recognizes GPI species, while human IgG is hardly recognized (FIG. 2).

The glycolipids containing the n-butanol phase obtained by in vivo labeling as described above were each spotted on a pre-processed Silica 60 HPTLC plate (10 x 20 cm, Merck), and the solvent system n-hexane / chloroform / methanol / Water / acetic acid (3: 10: 10: 2: 1).

The chromatogram was dried and scanned for radioactivity with a Berthold LB 2842 linear analyzer. ³ H- glucosamine, mannose, ³ H-, H- ³ H- ³ stearic acid or palmitic mountain any one metabolically labeled by Taki analyzes a n- butanol fraction of ZH detecting a constant six different peaks on TLC plate (Fig. 3). However, only three peaks of radioactivity were detected when the tagyzoids were labeled with ³ H-ethanolamine (Figure 3D), indicating that three of the six separate GPI species, peak fractions I, II, III only contains ethanolamine-phosphate molecules.

After the butanol phase containing ³ H-glucosamine-labeled glycolipids was dried, the cells were washed with 1 U PI-PLC (Bacillus cereus, product of Boehringer), 10 ul rabbit serum (source of PLD), 50 U PLA2 ) Or 10 μl buffer was dissolved in 100 mM Tris / HCl pH 7.4 containing 2 mM CaCl ₂ and 0.1% sodium deoxycholate. The sample was incubated at 37 占 폚 for 12 hours and heated for 5 minutes to inactivate the enzyme. The reaction mixture was partitioned between water and butanol, and the butanol phase was analyzed by TLC. All glycolipids were sensitive to GPI-PLD and PI-PLC, thus demonstrating their GPI structure. The susceptibility to PLA2 indicates that the lipid portion of the T. gondigalligil contains diacylglycerol.

Example 4

Immunoreactivity of several GPI species

To identify the peak fractions representing GPI species recognized by human IgM antibodies, each glycolipid was purified by purification TLC using glycolipids labeled metabolically with ³ H-glucosamine as tracer. The developed TLC-plate area corresponding to the peaks of radioactivity was scraped off, reextracted twice with 1.5 ml CMW, and fractionated between water and saturated n-butanol to remove residual silica. Several GPI species (indicating a material derived from 2 x 10 ⁷ Tachyzoites) were spotted onto a reinforced nitrocellulose membrane (Scheicher and Schuell) in 10 μl of butanol. The nitrocellulose strips were dried at 37 ° C and blocked overnight in 5% w / v skim milk in Tris buffered saline (15 mM Tris-HCl, pH 8, 140 mM NaCl, 0.05% w / v Tween 20). The blots were incubated with mAb T54E10 as well as sera from patients with toxoplasma. After washing, the blot is incubated with the appropriate anti-Ig-alkaline phosphatase or anti-Ig-peroxidase conjugate and the BCIP and NBT for alkaline phosphatase (Promega product), or peroxidase ECL, manufactured by Amersham) were detected by chemiluminescence detection method according to the supplier's protocol. As shown in Figures 4 and 5, human antibodies, more specifically human IgM and IgG antibodies, reacted constantly with peak fractions I, II, IV, and V. mAb T54E10 reacted with peak fractions I and II (Figure 4).

ELISA inhibition-binding studies were performed to demonstrate that some of the human IgM epitopes on GPI molecules were free ethanolamine-phosphate groups. That is, the GPI species of the n-butanol fraction was coated on the microtiter wells at a coating concentration equivalent to 10 ⁶ tachyzoites per well. As shown in Figure 6, only the binding of mAb T54E10 to n-butanol species was inhibited by ethanolamine-phosphate. Ethanolamine (10 mM) itself could inhibit the binding of mAb T54E10 by 50%. All other compounds tested failed to inhibit the binding of monoclonal antibodies. However, the binding of human anti-toxoplasma antibodies was not affected by any of the compounds tested.

Example 5

Structural Analysis of GPI Species Using Metabolically Labeled TLC Purified Glycolipids

To make an affinity headgroup, the glycolipids labeled with [ ³ H] glucosamine and dried by TLC purification were dried and resuspended in 400 μl of 0.1 M sodium acetate (pH 3.5) containing 0.25 M NaNO ₂ And incubated at room temperature for 4 hours. The reaction was stopped by adding 300 쨉 l of 0.4 M boric acid. This was top-fractionated with butanol / water and the lipid-free material was recovered from the aqueous phase. This material was reduced using NaBH ₄ and desalted against AG50W-X12 as previously described (Field & Menon, Lipid changes in protein, Hooper & Turner, Oxford, 155-190, 1992). The leader was resuspended in 100 μL of 50 mM NaAc (pH 4.5) containing 0.2 mM ZnCl ₂ , 0.02% sodium azide and 0.5 U Jack bean α-mannosidase (JBAM) or buffer, and incubated at 37 ° C. for 24 hours Respectively. The hydrophilic head of each glycolipid was analyzed by bio-gel P4-gel filtration before and after treatment with JBAM. The sample was mixed with 150 μl of partially hydrolyzed dextran (150 mg / ml, prepared according to Yamashita et al., Meth. Enzymol. 83, 105-126, 1982), equilibrated with 200 mM ammonium acetate and eluted Was loaded into a 140 x 1 cm bio-gel P4 column (BioRad). Standard detection reactions were performed by orcinol staining as described in the literature (Tomavo et al., 1992). Of the six different glycolipids, four sizes of hydrophilic heads (9.5, 8.5, 7 and 6 GU, respectively) were detectable. JBAM had broad specificity for all terminal unsubstituted α-D-mannose residues. The headers derived from glycolipids I, II and III did not respond to manocidase treatment as opposed to the head groups derived from glycolipids IV, V and VI, and this result is consistent with the labeling data with the terminal mannose of glycolipids I, II and III Lt; RTI ID = 0.0 > ethanolamine < / RTI > phosphate.

In order to isolate the core glycans of the GPI species, [ ³ H] glucosamine labeled and TLC purified glycolipids were dephosphorylated, thallamized and reduced by most conventional methods (Mayor et al., Method in Enzymol., 1 , 297-305, 1990). Briefly, the reaction was terminated by incubation at 48 ° C with aqueous HF at 0 ° C for 60 hours, dephosphorylation, and neutralization with frozen LiOH. The reaction mixture was desalted, deaminated and reduced as described above and finally dissolved in 1 ml of AG50W-X12 and 0.4 ml of Shellex 100 (Na ⁺ ), 0.2 ml of AG50W-X12 (H ⁺ ), 0.4 ml of AG3 -X4 (OH ^{-) -} ion in tandem and QAE- Sephadex (OH) - of 0.2㎖ and desalted on a column exchange and filtered to 0.25㎛ filter. The core glycans were analyzed by bio-gel P4 gel chromatography, Dionex HPAEC and lecithin affinity chromatography.

Radiolabeled core glycans were analyzed in Dionex bio-liquid chromatography (HPAEC). Samples were mixed with partially hydrolyzed dextran prior to injection into a Carbopaque PA1 column equilibrated with 100 mM NaOH. Elution was performed by using 100% Buffer A (100 mM NaOH) for 6 minutes followed by a linear increase of 0-30% Buffer B (100 mM NaOH, 0.25 M NaOAc) for 30 minutes at a flow rate of 1 ml / min. 0.4 ml fractions were collected. Cored glycans from all six glycolipids were simultaneously co-eluted using 3DU (dextran unit, where Glc ₃ ) standards. This elution position is clearly different from the 2.5DU elution position of the sample Man3-AHM core glycan, It is considered to be a substitute for glycans of Gondii GPI.

The radioactively labeled core glycans derived from TLC purified GPI were re-analyzed with Dionex-HPAEC after treatment with JBAM as described above and then with demineralized samples. The elution location of the major reaction product was 2.4 DU for all six glycolipids. This elution position is consistent with the limited susceptibility of glycans to JBAM, since it blocks non-mannose substituents attached to the triannosyl core (unconjugated core glycans are eluted as 0.9DU after JBAM treatment). To investigate substituent properties, monosaccharide analysis of radioactively labeled core glycans was performed. Core glycans derived from [ ³ H] glucosamine-labeled glycolipids II and III were dried and hydrolyzed with 4N HCl at 100 ° C for 4 hours. The cooled reaction mixture was dried and flash-evaporated twice with methanol 5% acetic acid and twice with methanol. The hydrolyzate was mixed with a monosaccharide standard mixture containing 10 nmoles of each monosaccharide, and analyzed with Dionex-HPAEC using equilibrated elution with 15 mM NaOH at a flow rate of 1 ml / min and 0.25 ml fractions were collected. The elution position of each monosaccharide standard was detected using a pulsed amperometric detector. Two radiolabeled monosaccharides were identified as anhydrous mannitol and galactosamine. Anhydrous mannitol is a derivative of the deaminated and reduced deacetylated glucose amine of GPI. The discovery of galactosamine suggests that N-acetyl galactosamine (GalNAc) is an additional component of core glycans of GPI.

To determine the presence of terminal GalNAc residues, the core glycans from all six glycolipids were incubated with [beta] -n-acetylhexosaminidase, an exoglycosidase that cleaves the terminal [beta] -linked N-acetylhexosamine Lt; / RTI > Briefly, the sample was resuspended in 100 μl of 50 mM sodium citrate pH 4.5 containing 0.5 U recombinant enzyme (New England BioLab), incubated for 12 hours at 37 ° C, heated, desalted, The cells were analyzed for Dionex-HPAEC described above. Glycolipids III and IV were sensitive to hexosaminidase treatment. Two fragments can be identified after treatment with HPAEC co-eluted with GalNAc (1DU) and Man3-AHM (2.5DU), suggesting the presence of N-acetyl galactosamine in these glycans. To show that GalNAc is a substitute blocking mannosidase activity, it was treated with hexosaminidase and JBAM. HPAEC analysis of glycans from glycolipids III and VI treated in this manner results in two peaks co-eluting with GalNAc and anhydrous mannitol showing the mannosidase sensitivity of the complete glycan after removal of the GalNAc residue. In contrast, core glycans derived from sugar lipids I, II, IV and V showed that n-acetyl galactosamine was present by monosaccharide analysis but was insensitive to hexosaminidase.

Analysis of coreglycan bio-gel P4-gel filtration showed the size heterogeneity of the core glycans. Coreglykans derived from glycolipids III and VI were co-eluted with the Man3GalNAc-AHM standard material form, rat brain Thy 1 antigen membrane attachment and 6GU standard (referred to as Coreglycan A). Glucan from hexaamyangiase insensitive glycoproteins I, II, IV and V was co-eluted with a 7GU standard (referred to as coreglycan B). These findings show the presence of additional substituents on the GalNAc moiety that steric hindrance to the enzymatic removal of GalNAc.

These data were performed by lectin-affinity-chromatography using lectin-specific Wistaria floribunda adhesion to terminal? 1,4-linked GalNAc. Lectin affinity chromatography was performed as previously described (Nakano et al., Arch. Biochem. Biophys. 311, 117-126, 1994). Briefly, radiolabeled core glycans derived from glycolipids II, III (core glycans A and B, respectively) and rat brain THY1 membrane fixation were dissolved in 50 mM Tris-HCl containing 0.02% sodium azide, pH 7.4 Lt; / RTI > Applied to a Floribunda adhesion agarose column (5 mg lectin / ml gel). Elution was carried out using 15 ml of the same buffer containing 100 mM of N-acetylgalactosamine as a specific inhibitor after using 25 ml of equilibrium buffer. Coreglycan A and Man3GalNAcAHM-standards were bound to the column and eluted with the specific inhibitor GalNAc (Figure 7), whereas coreglycan B did not exhibit affinity for lectin, indicating that there is an additional parental in these GalNAc species This shows that lectin can not be accessed by this.

Example 6

Identification of Glucose as a Component of Core Glycan B by an In Vitro Label Using a Tachyzoite Cell Glass System

An experimental approach to in vivo labeling can solve the in vivo labeling problem of Takizoites in often occurring cell cultures. These problems are mainly due to ineffective delivery of labels through the various membrane systems (host cell plasma membranes, metaplastic vacuoles and Tachyoid membranes) around the intracellular metabolism and / or excessive catabolism or epimerization of the labeled sugars, This results in poor and non-specific labeling. The in vitro labeling was carried out on T. It is used to show the glycosylation of Gondii GPI.

Tachyzoites were isolated from the infected BERO cell culture and purified from the host cell membrane by gel filtration as described above. Cellular glass systems for in vitro labeling were prepared by modification according to Masterson et al. (Cell 56, 739-800, 1989). Briefly, 2 × 10 ⁹ Takizoids were subjected to storage degradation in 375 μl of cold water containing 1 μg / ml leupeptin and 0.1 mM Tosyl Lysine Chloromethylketone (TLCK) for 5 min on ice, Dounce) homogenizer. The lysate was mixed with an equal volume of 2x concentrated culture buffer (50 mM NaHEPES pH 7.4, 25 mM KCl, 5 mM MgCl ₂ , 0.1 mM TLCK and 1 μg / ml of leupeptin) and homogenized again briefly. 75 [mu] l of Takizoit's cell membrane preparation representing 2 x 10 ⁸ Tachyzoites was added to each culture tube (containing pre-dried nucleotides, CoA and ATP) and cultured at 37 [deg.] C for 2 hours. The culture tubes contained 1mM ATP, CoA, UDP-GlcNAc, UDP-GalNAc and GDP-Man (for non-GDP-3H-Man labeling) and 2μCi GDP-3H-Man or 5μCi UDP-3H-Gluc. 2 ml of CM was added to terminate the culture and GPI extraction was carried out continuously as described above. Coreglycans can be prepared from glycolipids contained on butanol by dephosphorylation, desamination and reduction as described above. The resulting core glycans were analyzed by Dionex-HPAEC and aminopropyl-HPLC (Fig. 8). (See Example 5 for detailed experiments).

Ex vivo labeling with GDP-3H-Man results in the formation of a glycolipid comprising a core glycan containing Man3AHM and a core glycan A (Man3 (GalNAc) AHM) as described above (Tomavo et al., 1992) Room B is not synthesized under these conditions (Fig. 8D). In contrast, labeling with UDP-3H-Glc co-elutes coreglycan B (Figure 8H) to form an aminopropyl-HPLC peak that is not labeled with coreglycan A and Man3AHM.

Coreglycan B labeled with UDP-3H-Glc was purified by aminopropyl-HPLC. The purified labeled glycan was dried in Rectivial ^™ , and 200 μl of 2N trifluoroacetic acid was added and cultured at 100 ° C. for 4 hours to hydrolyze. The hydrolyzate was desalted and the Dionex-HPAEC monosaccharide analysis described above was performed. All radioactivity was co-eluted with glucose standards (Figure 9). This data shows that the additional substituent in coreglycan B is glucose. Stimulation experiments with unlabeled UDP-Glc were performed to confirm this observation. The cell glass system of the Tachyzoite membrane as described above was supplemented with 1 mM UDP-Glc and labeled with GDP-3H-Man. Analysis of the core glycans produced under these conditions is not related to the presence of Man3AHM and core glycan A, nor to the formation of core glycan B (Fig. 8F).

Example 7

Explanation of the complete structure of core glycans A and B

tea. The steps selected for the structural description of the Gondi GPI core glycan are as follows:

1. Extraction from multiple tachyzoites and separation of glycolipids by butanol / water phase partitioning,

2. preparing core glycans by dephosphorylation, deamination and reduction from a butanol phase containing all six glycolipids,

3. HPLC-Purification step for purification of core glycans A and B on amino-propyl,

4. Methanololysis / extensive analysis of glycans purified by GC, FAB-MS, GC-MS and NMR.

Since core glycans A and B co-elute on the Dionex-HPAEC column and dissolve incompletely in the bio-gel P4, a large amount of each glycan species was uniformly purified by separating the sample on aminopropyl-HPLC.

The core glycans were analyzed and purified using a Waters 510 HPLC system. To use the separation system, 50 μl of radioactively labeled core glycans in water was injected into the equilibrated Liquorsol-NH ₂ column (4.9 × 250 mm, Merck) and eluted with 28% water and 72% % Acetonitrile. 1 ml fractions were collected and the elution position of each glycan was confirmed by scintillation counting (Fig. 10A). Fractions containing peaks A and B were pooled and analyzed using bio-gel P4 gel chromatography (Figure 10E, G) and Dionex-HPAEC (Figure 10D, F). These experiments show that Peak A is identical to Core Glycan A (co-elution with a 6GU reference material on BioGel P4) and Peak B is identical to Core Glycan B (co-eluting with the 7GU standard).

To obtain a large amount of radiolabeled core glycans used as tracers in the purification of unlabeled material, chemical radioactivity labeling was performed. The glycolipids obtained from 2 x 10 ⁹ were dephosphorylated and deaminated as described above. The pH was adjusted to 10.5, and radioactivity labeling was performed by adding ₅ mCi NaB [ ³ H] H ₄ in 10 μl of 0.1 N NaOH and incubating for 30 minutes. Solid phase extraction on 1 ml Aminopropyl Sep Pak ^TM -cartridge (Millipore-Waters) was applied to remove radiochemical impurities. The cartridge was prewashed with a syringe with 8 mL of water, 2 mL of ethanol and 5 mL of butanol. The sample was taken in 40 μl of water, mixed with 2 ml of butanol, applied to a cartridge and eluted with 2 ml of butanol, 4 ml of butanol / ethanol (2: 1), 18 ml of ethanol and 3 ml of water . Purified radiolabeled core glycans were recovered from water. The material was analyzed in parallel with core glycans formed from GPI metabolically labeled by Dionex-HPAEC, bio-gel P4 and aminopropyl-HPLC. This analysis yielded the same pure radiolabeled material (1.5 x 10 < ⁷ > cpm) as the metabolically labeled glycolipids, starting with 2 nmoles of glycans.

For manufacturing purposes, 25 nmol core glycans were mixed with 50,000 cpm radio tracers in each operation. The core glycans from 1.5 x 10 ¹¹ tachyzoites were purified by HPLC to give approximately 30 nmol of glycan A and 150 nmol of glycan B. This material was further analyzed using monosaccharide analysis and different mass spectrometry and NMR techniques.

Methanol decomposition was performed as described above (Ferguson, GPI membrane anchors: Isolation and analysis, in Glycobiology. A practical approch, Fukuda and Kobata plate, IRL Press, Oxford University Press, Chapter 8, p349-383, 1993). Briefly, a sample containing core glycans A and B, respectively, derived from 2x10 < 10 ^> Tachyzoites was dried and resuspended in dry methanol containing 1N HCl and incubated at 85 [deg.] C for 24 hours. The cooled reaction mixture was neutralized with AgCO ₃ followed by addition of 25 μl of acetic anhydride and re-N-acetylation by incubation for 15 minutes. After centrifugation, the supernatant was dried in Speedback ^™ (Saban), and 40 μl of the newly prepared pyridine / hexamethyldisilazane / trimethylchlorosilane (Pierce) 5: 1: 1 was added and reacted for 30 minutes, . The derived monosaccharide was analyzed by performing Hewlett Packard 5890 gas chromatography with a 25 m x 0.32 mm fused silica CP-SIL-5 CF column.

The initial oven temperature of 130 [deg.] C was raised to 220 [deg.] C at a rate of 4 [deg.] C / min and detected by flame ionization.

In both samples, mannose, N-acetyl galactosamine and 2,5-anhydrous mannitol were detected at an approximate ratio of 3: 1: 1, indicating the presence of the trimannosyl core structure substituted by GalNAc. 2.8 mol of xylose per mol of GalNAc and 2 mol of glucose were identified in a sample containing coreglycan B and 1.3 mol of glucose was identified in a sample containing coreglycan A. [ Both sugars were frequently contaminated in the monosaccharide assay, but there is no difference in any other monosaccharide between A and B, suggesting xylose or glucose as a substitute for GalNAc present in coreglycan B. A mass spectrometer was used to solve this problem.

Prior to analysis by fast atom impact mass spectrometry (FAB-MS), coreglycan samples were obtained from Ciucanu & Kerek (Carbohyd. Res. 131,209, 1984) as described in Ferguson et al., Science 239, 753, Lt; / RTI > Briefly, the sample was dried and resuspended in 100 μl of dimethyl sulfoxide (Pierce) containing 120 mg / ml of powdered NaOH. After a certain agitation, 20 占 퐇 of methyliodine (Sigma) was added three times and incubated for 10 minutes after each addition. The reaction was quenched with sodium thiosulfate and the hypermethylated glycans were extracted with chloroform, washed with water and concentrated. Samples were resuspended in 5 μl of methanol and loaded onto a matrix of glycerol. Spectra were obtained using a Finnigan MAT 90 mass spectrometer adapted using a WATV Karim Gun (Wagner Arizen Technique) operated at 22 kV and 2 uA.

And FAB spectrum (Figure 12) recorded for the methylated core glycan A showed molecular ion m / z 1078.4 and each sodium adduct m / z 1100.4. The mass corresponds to the structural suggestion of Man-Man (GalNAc) Man-AHM for glycan A. Analysis of glycan B detected M ⁺ Na ⁺ ions at m / z 1282.7 and m / z 1304.5 for each M ⁺ Na ⁺ ion. Thus, the mass difference between hypermethylated glycans A and B is 204.1 D, which can accurately account for additional per capita differences on glycan B (Dell et al., Glycobiology, Fukuda & Kobata (eds) , 187-222, 1993). The ungluformized glycans were analyzed by MALDI-TOF mass spectrometry to confirm this (data not shown).

The core glycans A and B isolated and purified from 10 ¹¹ tachyzoites were subjected to nuclear magnetic resonance spectroscopy (NMR) to identify and define composition, anomer and binding pattern. A 2D homomorphic Hartmann-Haan (HOHAHA) spectrum was obtained by the method described by Marion et al., J. Jones et al., J. Phys. Lett .; Glycobiology, Fukuda & Kobata (eds.) Oxford, 221-242, . Mag. Reson. 85, 393-399, 1989), the 2D rotary flame nuclear overhauser effect (ROESY) is described in Bothner-By et al. Amer. Cem. Soc. 106, < RTI ID = 0.0 > 811-813. ≪ / RTI >

The 1D spectra obtained from core glycans A and B are shown in Fig. GPI membrane attachment of Bruce VSG (Ferguson et al., Science 239, 753-759, 1988) and rat brain THY1 (Homans et al., Nature 333, 269-272, 1988) and interpretation Respectively. For the core glycan A signal of both anomers from the 3? -Bonded mannose residues, anhydrous mannitol and one? -Bonded GalNAc were detected (FIG. 13). Coreglycan B showed additional α-anomer signals compared to saccharose and maltose identified as α-linked glucose. This finding identified additional proton and extended range using HOHAHA spectroscopy on coreglycan B. The ppm values of identified protons for core glycans A and B are summarized in Tables 1a and 1b.

Core Glycan A: Man3 Man2 Man1 AHM-ol GalNAc H-1 5.038 5.181 5.072 3.707 4.487 H-2 4.069 4.053 4.038 3.931 3.924 H-3 3.849 3.946 3.931 4.221 3.761 H-4 3.640 n.d. n.d. 4.159 3.940 H-5 n.d. n.d. n.d. 4.048 n.d. H-6 n.d. n.d. n.d. 3.736 n.d. NAc - - - - 2.075

Coreglycan B: Man3 Man2 Man1 AHM-ol GalNAc Glc H-1 5.038 5.185 5.075 3.685 4.542 4.936 H-2 4.070 4.052 4.029 3.968 3.983 3.551 H-3 3.848 3.955 3.918 4.223 3.857 3.814 H-4 3.639 3.72 3.79 4.163 4.039 3.464 H-5 n.d. n.d. n.d. 4.064 n.d. n.d. H-6 n.d. n.d. 3.76 3.752 n.d. n.d. NAc - - - - 2.073 -

A ROESY spectrometer was used in core glycan B to identify the carbon atom of each monosaccharide residue associated with the glycosidic linkage. Table 2 summarizes the relevant cross peaks.

A ROESY cross peak to identify glycosidic bonds in core glycan B Man 3 H-1 / Man 2 H-2 5,038 / 4,052 Man 2 H-1 / Man 1 H-6 5,185 / 3,76 Man 1 H-1 / AHMol H-4 5,075 / 4,163 GalNAc H-1 / Man 1 H-4 4,542 / 3,79 Glc H-1 / GalNAc H-4 4,936 / 4,039

These data are shown in Fig. Mannos skeleton of gondigliacans A and B is identical to the evolutionarily conserved structure Manα1,2-Manα1,6-Manα1,4-AHM described above (McConville & Ferguson, Biochem. J. 294, 305-324, 1993 ). GalNAc is β-1,4 linked to the first mannose (nearly close to AHM) and glucose is linked to GalNAc in α1,4 bond in coreglycan B. The structure of coreglycan A is similar to that of obesity-noctilized core glycans in the rat brain THY 1 membrane fixation (Homans et al., Nature 333, 269-272, 1988), while Coreglycan B is not yet disclosed in the literature Lt; RTI ID = 0.0 > GalNAc. ≪ / RTI >

Data obtained by the NMR spectrometer at the glycosidic bond sites was performed by the metallization analysis performed according to the previously published procedure (Ferguson, Science 239, 753-759, 1988). Briefly, 80 nmol of core glycan B was hypermethylated and acid hydrolyzed and the resulting hypermethylated monosaccharides were reduced and acetylated. After this process, partial hypermethylated alditol acetal (PMAA) was obtained having different degrees of methylation versus O-acetylation depending on the number and location of glycosidic bonds involved before hydrolysis. These PMAAs were separated and quantified using a gas chromatography-mass spectrometer (GC-MS). The total ion current chromatogram and the mass spectrum of each peak are shown in Fig. The four GC peaks were identified as PMAA (all terminated as mannose and glucose) with two terminal chitosan residues by GC retention time and mass spectra, a single hexose bonded to carbon 1 and 2, carbon 1,4 and 6 Lt; RTI ID = 0.0 > N-acetyl < / RTI > The resulting structure is also shown in Figure 14, consistent with data obtained by NMR and exoglycosidase.

tea. All the data obtained by structural analysis of Gondigans GPI is consistent with the structure shown in FIG.

Structural representations of the glycan core structures A and B are shown in Fig.

Example 8

Chemical synthesis and conjugation of sugar conjugate structure of core glycans A and B

synthesis:

Compounds A and B as well as their analogs can also be synthesized by various routes. An example of the synthesis process of octagonal 13 is summarized. In analogues of compound B, the non-essential anhydrous mannitol unit was substituted with a 3-aminopropyl bond handle to facilitate conjugation of the macromolecular carrier to the saccharide. In addition, a binding handle having different chain lengths or containing functional groups (e.g., carboxyl groups, thiols or halides) suitable for other bonding purposes can be used. The synthesis is irrelevant to mannose unit 1 and can be readily accessed by standard procedures from commercial ethyl 1-thio-α-D-mannosyl pyranoside (eg, p-methanesulfonyl chloride in the presence of p- 4,6-Op-methoxybenzylidene group with methoxybenzaldehyde dimethylacetal followed by benzylation of the resulting 2,3 diol with benzyl bromide and sodium hydride. (Veeneman et al., 1990, Tetrahedron Lett., 31, 1331) in the presence of N-iodosuccinimide (NIS) and catalytic trifluoromethanesulfonic acid (TfOH), compound 1 and 3-N- benzyloxycarbonylamino - propan-1-ol the compound 2 is formed by the reaction of (.. etc. Berntsson, 1977, Acta Pharm Suec, 14, 229). Compound 2 was treated with dry CF ₃ COOH in NaBH ₃ CN and (DMF) to selectively remove the 6-Op-methoxybenzyl (Mbn) fraction from (Johansson et al., 1984, J. Chem. Soc. Perkin Trans. 3 < / RTI > 3 and N-Trichloroacetyl- (TCA), prepared in a manner similar to that reported for glucosamine in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) (Blatter et al., 1994, Carbohydrate Res., 260, 189) Coupling with galactose amine derivative 4 yields disaccharide 5 . Conversion of trichloroacetyl (TCA) groups to acetyl groups was carried out using BuSnH and 2,2'-azobis (2-methylpropionitrile) (ABIN). The saponification of the three O-acetates can be selectively benzylated using the following two-step procedure by allowing access to the corresponding triols, 4-OH and 6-OH functional groups: 1) treatment with excess Bu ₂ SnO ₂ and 2) Reaction with BnBr in the presence of CsF. The generated derivatives NIS and Catalytic TfOH glucose unit 6 in the presence of (such as Veeneman, 1990, Tetrahedron Lett, 31 , 1331); by coupling in (Veeneman GH, 1991, Thesis Leiden University) and 3-OH functional group trimer 7 ≪ / RTI > Compound 8 was obtained by treating 7 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (Oikawa et al., 1982, Tetrahedron Lett., 23, 885). 8 was reacted with mannose unit 9 (Elie et al., Tetrahedron, 46, 8243) in the presence of NIS-TfOH to give tetramer 10 which was saponified with NaOMe to remove 2-O-benzyl to give 11 . Then, 11 is condensed with 9 to induce pentasaccharide 12 . Finally, the protecting group is removed by hydrogenation in the presence of 1) NaOMe and 2) 10% Pd to obtain the target compound 13 .

join:

Sucrose 13 can be readily coupled to a protein carrier as previously described (Evenberg et al., 1992, J. Infect. Dis., 165, S152-s155). First, 13 was reacted with hydroxysuccinimide ester of commercially available acetylthioacetic acid to obtain 14. Compound 14 is then coupled with a bromoacylated protein (e. G., BSA, KLH, Tentanus toxin) in the presence of hydroxylamine to yield conjugate 15 . In addition, 13 can be directly coupled with the protein in the presence of glutaraldialdehyde to obtain a saccharide-protein conjugate.

The above synthesis and bonding are illustrated in Fig. 17, which shows the reaction outline.

Claims (22)

A sugar conjugate comprising a glycan core structure represented by the following formula (1): < EMI ID = Formula 1 R 1 = hydrogen, -PO ₃ -CH ₂ -CH ₂ -NH ₂ (ethanolamine-phosphate) or -PO ₃ -CH ₂ -CH ₂ -NH-X, wherein X = Gondii-specific antigens; (1-4) -O-anhydrous mannitol or [alpha] (1-4) -O-glucoseamine, optionally with an alpha (1-6) -linkage to the inositol moiety of a phosphatidyl inositol membrane adherend ; R3 = monosaccharide moiety; R4 = hydrogen or -L-R5, wherein L = 2 is an organic radical; R5 = a functional group capable of coupling to the carrier. 2. The saccharide conjugate according to claim 1, wherein the monosaccharide portion is a hexose saccharide. The saccharide conjugate according to claim 2, wherein the saccharified sugar portion is a glucose moiety. The saccharide conjugate according to claim 3, wherein the glucose moiety is bonded to the GalNAc moiety by an? -1,4-glycoside bond. The sugar conjugate according to any one of claims 1 to 4, wherein the sugar conjugate comprises a structure represented by the following formula (2): In the above formula, X = T. Gondii specific antigen. The method according to any one of claims 1 to 5, wherein the sugar conjugate is tea. The rupture stage of the Konditaki's zeit membrane and Extracting the resulting tachyzoite suspension with a mixture of chloroform / methanol / water (C / M / W: 1/1 / 0.3 v / v) Wherein the saccharide conjugate is obtained from a gonadotropin. 7. Human monoclonal antibody according to any one of claims 1 to 6, wherein said conjugate is a human monoclonal antibody produced by an infinite proliferation cell line deposited at accession numbers 95090608 and 95090609 to the European animal cell culture harvest collection (ECACC) Wherein the antibody is immunoreactive with the antibody. A tea having immunoreactivity with the sugar conjugate according to any one of claims 1 to 7. Antibodies induced by conjugates of Gondigans. 9. The antibody of claim 8, wherein said antibody is a monoclonal antibody. Recognized by the monoclonal antibody TOX.HuOT-1 or TOX.HuOT-2 produced by the hybridoma cell line deposited at Accession No. 95090608 and 95090609 to the European animal cell culture collection (ECACC) And T. A monoclonal antibody that binds to an epitope present in a conjugate of the Gondii. Monoclonal antibody TOX.HuOT-1 or TOX.HuOT-2 produced by a hybridoma cell line deposited at Accession No. 95090608 and 95090609 to the European animal cell culture collection (ECACC) of Fortton Down, England, respectively. 12. An infinite proliferative cell line capable of producing the monoclonal antibody according to any one of claims 9 to 11. Infinite proliferation cell lines deposited at accession numbers 95090608 and 95090609 to the European animal cell culture collection (ECACC) of Fortton Down, England, respectively. An immunochemical reagent comprising the sugar conjugate according to any one of claims 1 to 7. An immunochemical reagent comprising an antibody according to any one of claims 8 to 11. Characterized in that the test fluid is contacted with the antibody of any one of claims 9 to 11 and then the presence of the immune complex is detected. Detection method of Gondi. 14. A test fluid in a test fluid characterized in that the immunochemical reagent according to claim 14 is contacted with a test fluid and the presence of the immunocomplex formed in the test fluid is detected. A method for detecting an antibody directed against Gondii. 16. An immunochemical reagent according to claim 15 is contacted with a test fluid, Contacting the induced antibody against Gondii to detect the presence of the formed immune complex. Detection method of Gondi. 14. A kit comprising an immunochemical reagent according to claim 14. Test kit for detecting Gondii infection. 15. A method of treating a tumor comprising administering to a mammal a therapeutically effective amount of an immunochemical reagent according to claim 15. Test kit for detecting Gondii infection. A specific inhibitor for inhibiting biosynthesis of the sugar conjugate according to any one of claims 1 to 7. A therapeutic agent for inhibiting the replication of a gonadal organism. Wherein the surface expression of the GPI-fixed antigen is blocked or inhibited by the therapeutic agent of claim 21. Production method of Gonadal live vaccine.